T cell receptor (TCR) ligation induces intracellular signaling cascades including the Ca2+-NFAT (nuclear factor of activated T cells) and MAPK (mitogen-activated protein kinase) pathways that play crucial roles in activation and differentiation of T cells. Defects in these pathways lead to immune deficiency or hypersensitive immune responses in humans and mice. The small GTPases, Ras, Rac and Rho are known to play a central role in activation of TCR signaling pathways and are therapeutic targets in autoimmune diseases. These small GTPases are recruited into the immunological synapse (IS), the interface between an antigen-presenting cell and a T cell via interactions with signaling adaptors and activate the downstream MAPK pathway. However, in contrast to Ras, Rac and Rho GTPases, little is known about the role of more than 60 members of the Rab GTPase family in T cell activation. Our long-term goal is to uncover the role of Rab GTPases in TCR signaling to identify novel therapeutic targets to alleviate autoimmunity. We have recently identified a novel Rab GTPase, CRACR2A GTPase that plays a key role in TCR signaling. Our preliminary data show that CRACR2A GTPase is a lymphocyte-specific large Rab GTPase with many functional domains in contrast to small GTPases. We found that CRACR2A GTPase translocates into the IS to activate the downstream JNK (c- Jun N-terminal kinase) MAPK pathway. Furthermore, it is highly expressed in TH1 cells and plays an important role in expression of IFN-? and T-bet, and thus in autoimmunity. Our central hypothesis is that CRACR2A GTPase is recruited into the IS via protein interactions and regulates the JNK pathway to drive TH1 differentiation. The objective of this proposal is to identify 1) interacting partners, 2) GTP binding and prenylation-mediated regulation, and 3) the physiological role of CRACR2A GTPase in T cells using high resolution imaging techniques, genomics and conditionally targeted knockout mice. CRACR2A GTPase is the first example of a large molecule containing the small Rab GTPase domain. We expect to reveal a hitherto unknown mechanism of regulation of the JNK pathway in T cell differentiation. We are confident that we will also identify a unique signaling function of Rab GTPases, which are otherwise known to mediate only intracellular protein trafficking. These mechanistic studies will have significant impact on therapeutic exploitation of this novel GTPase to treat autoimmune diseases.